A lack of sufficient quantities of genomic DNA for genomic analysis can be overcome by global amplification of all DNA within a sample (whole genome amplification). The REPLI-g FFPE Kit consists of DNA Polymerase, novel buffers, and reagents that enable efficient whole genome amplification from formalin-fixed and paraffin embedded (FFPE) tissue without the requirement for prior DNA purification. After lysis of the tissue section, the DNA is processed so that fragmented DNA is ligated. The long DNA strands created are amplified using proven REPLI-g technology.

The REPLI-g FFPE Kit is intended for molecular biology applications. This product is not intended for the diagnosis, prevention, or treatment of a disease.

Product Details

Reliable microsatellite analysis.

DNA from a 3.5-year-old FFPE carcinoma tissue sample was analyzed at a single microsatellite locus prior to and after DNA amplification using the REPLI-g FFPE Kit. Identical results were obtained for the original and REPLI-g amplified genomic DNA. (Data kindly provided by Hartmut Schmidt, Gerhard Domagk Institute for Pathology, Münster, Germany.)

High-molecular-weight ligated DNA.

DNA from different FFPE carcinoma tissues after (1a–5a) and before (1b–5b) amplification using the REPLI-g FFPE Kit was run on an agarose gel. The REPLI-g FFPE ligation procedure results in the formation of high-molecular-weight DNA. (Data kindly provided by Hartmut Schmidt, Gerhard Domagk Institute for Pathology, Münster, Germany.)

REPLI-g FFPE principle.

The REPLI-g FFPE procedure involves a novel DNA processing reaction that prepares and ligates fragmented DNA. This ligated DNA is then used in MDA-based whole genome amplification.

REPLI-g FFPE procedure.

Formalin fixation and paraffin embedding cause DNA fragmentation. During the REPLI-g FFPE procedure, these DNA fragments are randomly ligated and then amplified using multiple displacement amplification. As indicated, DNA fragments (containing different loci [L1–L4]) are not assembled in the original order. However, the different loci are equally amplified and can be detected in downstream genotyping applications.

Performance

For the two protocols with different amplification times, typical yields per 50 µl reaction with standard quality templates are up to 10 µg after 2-hour amplification (standard reaction) and up to 40 µg after 8-hour amplification (high-yield reaction).

Principle

The availability of sufficient quantities of genomic DNA for genomic analysis is often lacking. Whole genome amplification (WGA) overcomes this limitation by global amplification of all DNA within a sample, providing sufficient quantities to perform all analyses on the same DNA sample.

Genotyping of formalin-fixed, paraffin-embedded (FFPE) tissue samples enables morphological tissue changes to be directly linked to specific genome mutations. However, formalin fixation causes irreversible damage to the DNA, resulting in fragmented DNA that is cross-linked to other biomolecules within the sample. In addition, the limited amount of DNA that can be extracted from FFPE tissue samples is only sufficient for a few analyses.

The REPLI-g FFPE protocol allows the amplification of purified DNA from FFPE tissue samples or DNA direct from FFPE tissue samples without prior DNA purification. After lysis of the tissue section, the DNA is processed using novel buffers and enzymes that ligate fragmented DNA (see flowchart "REPLI-g FFPE procedure"and figure "High-molecular-weight ligated DNA"). The long DNA strands created by the ligation reaction are amplified using proven REPLI-g technology. Once amplified, the DNA is suitable for immediate use in most downstream genotyping assays without further purification.

DNA amplified using the REPLI-g FFPE procedure is highly suited for use in real-time PCR (e.g., using QuantiFast Kits) and end-point PCR (e.g., using the QIAGEN Fast Cycling PCR Kit), with a PCR amplicon preferably smaller than the average fragment size of the starting template. Further applications are microsatellite analysis (see figure "Reliable microsatellite analysis") and SNP genotyping (REPLI-g FFPE amplified DNA may not be suited for genotyping methods that require restriction digestion for DNA labeling).

Applications

DNA amplified using the REPLI-g FFPE procedure is highly suited for use in: